Full-length, mature platelet-endothelial cell adhesion molecule-1's (PECAM-1's) are glycosylated proteins with 711 amino acids and a molecular weight of approximately 130 kilodaltons. The proteins are members of the immunoglobulin superfamily. They are expressed on platelets, at the intercellular junctions of resting endothelial cells, and on circulating monocytes, granulocytes, and certain subsets of T-cells. Newman et al. (I)(1990) Science 247, 1219-1222; Muller, et al. (I)(1989) J. Exp. Med. 170, 399-414; Albelda, et al. (I)(1990) J. Cell. Biol. 110, 1227-1237; Ashman and Aylett (1991) Tissue Antigens 38, 208-212.
From a molecular cloning study, it is known that PECAM-1's have 6 extracellular Ig-like domains, a short transmembrane region, and a relatively long 118 amino acid (aa) cytoplasmic tail containing multiple potential sites for phosphorylation, lipid modification, and other post-translational modifications. Newman et al.(I), supra, and Newman, U.S. Pat. No. 5,264,554 (the '554 Patent), which is incorporated in its entirety herein by reference.
Three full-length, mature PECAM-1's have been found. One of these, designated herein as "form 1," has the amino acid sequence shown in FIG. 1 of the '554 Patent. The amino acid sequences of the other two are the same as the amino acid sequence of form 1 except that in one of them, designated herein as "form 2," which is the one for which portions of the amino acid sequence are provided in SEQ ID NO:3 through SEQ ID NO:11 hereinbelow, there is an asparagine rather than a serine at amino acid position 536, due to a change from a 2'-deoxyguanylate to a 2'-deoxyadenylate at nucleotide position 1829 in the cDNA sequence in FIG. 1 of the '554 Patent (which corresponds to nucleotide position 196 in SEQ ID NO:3 hereinbelow), Stockinger et al. (1990) J. Immunol. 145, 3889-3897, and in the other, designated herein as "form 2," there is an isoleucine rather than an asparagine at amino acid position 88 (resulting from a change from a 2'-deoxyadenylate to a 2'-deoxythymidylate at nucleotide position 485 in the cDNA of FIG. 1 of the '554 Patent), an aspartic acid rather than an asparagine at amino acid position 124 (resulting from a change from a 2'-deoxyadenylate to a 2'-deoxyguanylate at nucleotide position 553 in the cDNA of FIG. 1 of the '554 Patent), a methionine rather than an isoleucine at amino acid position 348 (resulting from a change from a 2'-deoxyadenylate to a 2'-deoxyguanylate at nucleotide position 1266 in the cDNA of FIG. 1 of the '554 Patent), and a valine rather than an aspartic acid at amino acid position 364 (resulting from a change from a 2'-deoxyadenylate to a 2'-deoxythymidylate at position 1313 in the cDNA of FIG. 1 of the '554 Patent), Zehnder et al. (1992) J. Biol. Chem. 267, 5243-5249. In addition, at several nucleotide positions in the cDNA's for PECAM-1's, silent substitutions (substitutions not resulting in amino acid changes) have been found. With reference to the cDNA sequence in FIG. 1 of the '554 Patent, such silent substitutions have been found at positions 514, 1593, and 2149 (which corresponds to nucleotide position 38 in SEQ ID NOS: 11 and 12 hereinbelow) in the amino acid-coding region and, in the 3'-untranslated region, position 2416 (which corresponds to nucleotide position 108 in SEQ ID NOS: 19 and 20 hereinbelow). See Newman et al.(I), supra; Stockinger et al., supra; Zehnder et al., supra; and the '554 Patent. Apparently, then, a number of nearly identical alleles of PECAM-1 genomic DNA exist in the human gene pool.
No polymorph of a PECAM-1 has been found with an amino acid substitution in the cytoplasmic domain, the amino acids at positions 594-711.
A soluble form of a PECAM-1, with a molecular weight between about 6,000 and 9,000 daltons less than that of a full length, mature PECAM-1 has been identified. Goldberger et al., Blood 80, 266a (1992).
PECAM-1's are important mediators of platelet-platelet, platelet-leukocyte, and platelet-endothelial cell interactions involved in platelet aggregation, development of atherosclerotic plaque, and development of thrombi as a result of vascular trauma, as may be caused, for example, by angioplasty or similar processes. PECAM-1's are also involved in leukocyte-endothelial cell interactions involved in processes such as transendothelial cell migration and related phenomena such as inflammation. Muller et al. (II) (1993) J. Exp. Med. 178, 449-460 and Vaporciyan et al. (1993) Science 262, 1580-1582 describe the use of PECAM-1 specific antibodies to interfere with neutrophil recruitment and transendothelial migration. The mechanisms by which PECAM-1's mediate these cellular interactions are complex, as PECAM-1's can interact both homophilically (a PECAM-1 molecule binding to a PECAM-1 molecule), Albelda et al. (II)(1991) J. Cell. Biol. 114, 1059-1068, as well as heterophilically (a PECAM-1 molecule binding to a molecule other than a PECAM-1 molecule), Muller et al. (III)(1992) J. Exp. Med. 175, 1401-1404 and DeLisser et al. (1993) J. Biol. Chem. 268, 16037-16046, to carry out its adhesive functions.
The cytoplasmic domain of a PECAM-1 molecule is the 118 C-terminal amino acids, amino acids 594-711, in the mature molecule. This domain plays an important role in regulating the adhesive properties of a PECAM-1. Removal of C-terminal portions in recombinant PECAM-1 constructs has been found to convert the molecule from heterophillic to homophilic ligand-binding specificity, DeLisser et al. (II)(1994) J.Cell.Biol. 124, 195-203. The cytoplasmic domain has sites for phosphorylation and lipid modification and interacts with the cytoskeleton. N ewman et al. (II) (1992) J. Cell. Biol. 119, 239-246 (1992); Zehnder et al., supra. Modifications in the cytoplasmic domain affect not only the adhesive properties of a PECAM-1's extracellular domain but also its subcellular distribution, interactions with intracellular signalling molecules, and ability to participate in intercellular signal transduction.